菸鹼酸羥肟酸抑制酪胺酸酶及調控黑色素生成之機制探討

Nicotinic acid hydroxamate: a potent tyrosinase inhibitor and mechanisms on down-regulation of melanin synthesis

林音秀(Lin Yin-Shiou)

指導教授 ： 侯文琪

臺北醫學大學/藥學院/藥學系(博士班)/博士(2012年)

摘要

本研究中使用四個菸鹼酸衍生物：菸鹼醯胺、菸酸甲酯、菸鹼酸羥肟酸、碘化甲基菸鹼酸羥肟酸及正向對照組麴酸，來觀察抑制酪胺酸酶及抗氧化的活性，菸鹼酸衍生物顯示具有抑制酪胺酸酶的活性，按順序為菸鹼酸羥肟酸>麴酸>甲基菸鹼酸羥肟酸碘鹽>菸鹼酸甲酯>菸鹼醯胺。其中菸鹼酸羥肟酸對於酪胺酸酶中的單酚酶活性及多酚酶活性的半抑制濃度分別為2 µM 及 1 µM，酵素抑制模式分別為混合型抑制以及不競爭抑制；在抗氧化能力方面菸鹼酸羥肟酸及甲基菸鹼酸羥酸碘鹽具有濃度依賴清除DPPH自由基能力，半抑制濃度個別為65.81 μΜ及125 μΜ，菸鹼酸羥肟胺酸及甲基菸鹼酸羥肟酸碘鹽也具有清除氫氧自由基及抑制低密度脂蛋白過氧化；進一步觀察菸鹼酸羥肟酸對於人類黑色素細胞及老鼠黑色素細胞瘤B16F10的抑制黑色素生成機制，發現菸鹼酸羥肟酸對於人類黑色素細胞及B16F10細胞的細胞萃取物中酪胺酸酶活性具有濃度依賴性抑制作用。以老鼠黑色素細胞瘤B16F10細胞做後續機制探討，在環磷酸腺苷(cAMP)的刺激劑(forskolin, isobutylmethylxanthine, and α-melanocyte stimulating hormone)的作用下，菸鹼酸羥肟酸處理的B16F10的黑色素含量及酪胺酸酶活性明顯地低於未經處理的細胞。在抑制黑色素生成的訊息傳遞訊號的研究顯示，菸鹼酸羥肟酸能降低酪胺酸酶、酪胺酸相關蛋白-1及MITF轉錄因子蛋白質的表現。處理MEK 的抑制劑PD98059 或phosphatidylinositol 3-kinase的抑制劑LY294002皆能明顯增加菸鹼酸羥肟酸處理B16F10後之黑色素的含量；另外，處理菸鹼酸羥肟酸後增加了MEK/extracellular signal-regulated kinases (ERK)、protein kinase B (Akt)及glycogen synthase kinase (GSK)-3β的磷酸化。因此推測可能經由磷酸化的訊息調控了MITF轉錄因子的表現，而降低了酪胺酸酶及TRP-1的表現，這些結果顯示菸鹼酸羥肟酸，為一個有效酪胺酸酶抑制劑及抑制黑色素的活性化合物。文獻指出菸鹼酸羥肟酸在Ames test下具有增加回復突變菌株數量，因此使用CD-1 (ICR)老鼠進行二十週2階段皮膚致癌性實驗，以評估菸鹼酸羥肟酸是否具有致癌的特性，結果顯示菸鹼酸羥肟酸在2階段皮膚致癌性實驗中不會扮演腫瘤起始物及促進物，這些結果顯示菸鹼酸羥肟酸及甲基菸鹼酸羥肟酸碘鹽具有潛力成為化妝品添加物。

關鍵字

酪胺酸酶菸鹼酸羥肟酸 ； 黑色素生成

並列摘要

In this study, the 4 nicotinic acid derivatives, namely, nicotinamide, methyl nicotinate, nicotinic acid hydroxamate (NAH), N-methyl nicotinic acid hydroxamate iodide (NAH-M), and kojic acid (positive control), were used to investigate the tyrosinase inhibitory and antioxidant activities. Among these, the nicotinic acid derivatives showed tyrosinase inhibitory activity in the order of NAH > kojic acid > NAH-M > methyl nicotinate > nicotinamide. The concentrations of half-inhibition (IC50) of NAH against monophenolase and diphenolase activity were 2 µM and 1 µM, respectively; the inhibition mode was the mixed-type in the former and uncompetitive type in the latter. In the antioxidant activity assay, NAH and NAH-M but not nicotinamide or methyl nicotinate, showed dose-dependent DPPH radical scavenging activity, and the IC50 values were 65.81 μΜ and 125 μΜ, respectively. NAH and NAH-M also showed hydroxyl radical scavenging activity and anti-low-density-lipoprotein peroxidation. Mechanism of NAH in depigmentation effect of human epidermal melanocytes and B16F10 cells was further investigated. The NAH was found to show dose-dependent inhibition of tyrosinase activity in cell extracts of human epidermal melanocytes and murine melanoma B16F10 cells. Therefore, the B16F10 cells were used for investigations of antipigmental mechanisms. In the presence of cyclic adenosine monophosphate (cAMP) elevators (forskolin, isobutylmethylxanthine, and α-melanocyte stimulating hormone), the melanin content and tyrosinase activity in the NAH-treated B16F10 cells were significantly lower than those in the untreated cells. In the studies on signaling pathways for anti-melanogenesis, NAH-treated B16F10 cells showed a decrease in the expression levels of tyrosinase, tyrosinase-related protein-1 (TRP-1), and microphthalmia-associated transcription factor (MITF). Pretreatment with PD98059 (MEK-specific inhibitor) or LY294002 (phosphatidylinositol 3-kinase [PI3K] inhibitor) markedly increased the melanin content in B16F10 cells and NAH-treated B16F10 cells. Furthermore, the levels of phosphorylation in MEK/extracellular signal-regulated kinases (ERK), protein kinase B (Akt), and glycogen synthase kinase (GSK)-3β pathways were high in NAH-treated cells; high levels of phosphorylation may down-regulate MITF expression and decrease tyrosinase and TRP-1 expressions. These results suggest that NAH may be an effective tyrosinase inhibitor and act as an active component in the inhibition of melanogenesis. In literatures, it was reported that NAH increased the revertant numbers in Ames test, therefore the carcinogenic properties of NAH were assessed by performing two-stage skin carcinogenesis experiments in CD-1 (ICR) mice for 20 weeks, and the results revealed that NAH did not act as an initiator or a promoter of two-stage skin carcinogenesis. These results indicate that NAH and NAH-M have the potential to be useful in cosmetics.

並列關鍵字

tyrosinase ； nicotinic acid hydroxamic acid ； melanogenesis

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